Good morning. I've been trying to detect a low copy bacterial target using TaqMan Universal PCR Master Mix and specific primers/probe. Unfortunately, I only have about a 50% detection rate for known positives, and my threshold cycles are consistently high (36-37 out of 40 total cycles). Increasing primer concentration does not seem to help. On the other hand, increasing template volume has a moderate effect, making detection consistent and very gradually decreasing Ct number. The problem is I'm already working with limited available template volume.
I attempted to prepare a separate "pre-amplification reaction," where I would first amplify a larger sequence containing my target (using primers flanking my TaqMan primers and AmpliTaq Gold 360 master mix). I then took the product from this reaction and used it as template for my TaqMan reaction (similar to a nested PCR). This has not worked yet either.
Does anyone have any recommendations for 1) a low copy target just using TaqMan reagents or 2) a "nested PCR" scheme using them? Thank you in advance.
Jose,
A few questions for clarity. What type of sample are you using? How are you extracting DNA? What is your analytical sensitivity of your primers/probe?
The reason I ask is that we too also work with a low copy bacterium and our primers/probe are very sensitive to small amounts of target. We have found our biggest area of concern is sample processing. Typically we have to concentrate our eluted DNA with another spin kit (Zymo Clean & Concentrate) to increase detection by real-time PCR.
Hello Noah,
Thank you for your input. My samples are swabs. These swabs were brushed against animals to diagnose them for the presence of skin bacteria. I'm using a modified protocol for the extraction for Gram+ bacteria using the QIAamp cador Pathogen Mini kit and pathogen lysis tubes. Unfortunately, I have no numbers for the analytical sensitivity of my oligos, other than to say they're from good published literature.
You might be right and I might have to take it a step back, though. Concentrating my extracts is something I hadn't tried yet.
Hey Jose,
You mention that they are from known positive animals, have you plated a swab to show growth of your Gram + bug of choice? Another potential issue might be the pathogen lysis tubes (beads I assume) and shearing of DNA. We have found that sharp particles (like garnet) in homogenization tubes can potentially shear DNA, if you have low copy number and it shears nears your target, that could possibly explain it but probably only a one-off and not a consistent problem.
I assume you have done NanoDrop or Qubit to verify that your extraction produced DNA. Might want to think about a spiked internal amplification control to show that your QIAamp is extracting. I don't think that skin swabs would be ripe with inhibitors so you can probably check that one off.
You might want to streak out a swab, grow up the bacterium of choice and run in on the primers/probe to verify those are working. If you want to get an analytical sensitivity to see just how low you can go with the oligos sets, I would suggest ordering Gblocks from IDTDNA of your amplicon plus 50 bp up and down stream of your primers. Another potential issue (if your positive control also fails to amplify) is that you have a bad order or primers or probe and you just need to re-order. I have been told the fail rate on the oligos can be around 10%.
Hello Noah,
To answer your first question, no I have not. However, the known positive swabs do come from clinically-diagnosed animals. The reason why we're trying to develop a qPCR-based assay is because microbiology (i.e. plating) has yielded poor results; the bacterium is a slow grower and mostly gets out-competed by a number of other species.
That being said, I do have a bacterial pellet sample that I extracted using my current method. I used this sample to test the primers I ordered, so I know they work (with this optimal sample type, that is).
Thank you for the brainstorming, though. I think I will definitely review/refine my extraction method based on your comments.
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